Comparison of fidaxomicin, thuricin CD, vancomycin and nisin highlights the narrow spectrum nature of thuricin CD.

Vancomycin and metronidazole are commonly used treatments for Clostridioides difficile infection (CDI). However, these antibiotics have been associated with high levels of relapse in patients. Fidaxomicin is a new treatment for CDI that is described as a narrow spectrum antibiotic that is minimally active on the commensal bacteria of the gut microbiome. The aim of this study was to compare the effect of fidaxomicin on the human gut microbiome with a number of narrow (thuricin CD) and broad spectrum (vancomycin and nisin) antimicrobials. The spectrum of activity of each antimicrobial was tested against 47 bacterial strains by well-diffusion assay. Minimum inhibitory concentrations (MICs) were calculated against a select number of these strains. Further, a pooled fecal slurry of 6 donors was prepared and incubated for 24 h with 100 µM of each antimicrobial in a mini-fermentation system together with a no-treatment control. Fidaxomicin, vancomycin, and nisin were active against most gram positive bacteria tested in vitro, although fidaxomicin and vancomycin produced larger zones of inhibition compared to nisin. In contrast, the antimicrobial activity of thuricin CD was specific to C. difficile and some Bacillus spp. The MICs showed similar results. Thuricin CD exhibited low MICs (<3.1 µg/mL) for C. difficile and Bacillus firmus, whereas fidaxomicin, vancomycin, and nisin demonstrated lower MICs for all other strains tested when compared to thuricin CD. The narrow spectrum of thuricin CD was also observed in the gut model system. We conclude that the spectrum of activity of fidaxomicin is comparable to that of the broad-spectrum antibiotic vancomycin in vitro and the broad spectrum bacteriocin nisin in a complex community.

Nisin variants from Streptococcus and Staphylococcus successfully express in NZ9800.

AIMS: Increases in antimicrobial resistance have meant that the antimicrobial potential of lantibiotics is now being investigated irrespective of the nature of the producing organism. The aim of this study was to investigate whether natural nisin variants produced by non-Generally Recognized as Safe (GRAS) strains, such as nisin H, nisin J and nisin P, could be expressed in a well-characterized GRAS host. METHODS AND RESULTS: This study involved cloning the nisin A promoter and leader sequence fused to nisin H, nisin J or nisin P structural gene sequences originally produced by Streptococcus hyointestinalis DPC 6484, Staphylococcus capitis APC 2923 and Streptococcus agalactiae DPC 7040, respectively. This resulted in their expression in Lactococcus lactis NZ9800, a genetically modified strain that does not produce nisin A. CONCLUSIONS: Induction of the nisin controlled gene expression system demonstrates that these three nisin variants could be acted on by nisin A machinery provided by the host strain. SIGNIFICANCE AND IMPACT OF THE STUDY: Describes the first successful heterologous production of three natural nisin variants by a GRAS strain, and demonstrates how such systems could be harnessed not only for lantibiotic production but also in the expansion of their structural diversity and development for use as future biotherapeutics.

Molecular surveillance of measles and rubella in the WHO European Region: new challenges in the elimination phase.

BACKGROUND: The WHO European Region (EUR) has adopted the goal of eliminating measles and rubella but individual countries perform differently in achieving this goal. Measles virus spread across the EUR by mobile groups has recently led to large outbreaks in the insufficiently vaccinated resident population. As an instrument for monitoring the elimination process and verifying the interruption of endemic virus transmission, molecular surveillance has to provide valid and representative data. Irrespective of the country's specific situation, it is required to ensure the functionality of the laboratory surveillance that is supported by the WHO Global Measles and Rubella Laboratory Network. AIMS: To investigate whether the molecular surveillance in the EUR is adequate for the challenges in the elimination phase, we addressed the quality assurance of molecular data, the continuity and intensity of molecular monitoring, and the analysis of transmission chains. SOURCES: Published articles, the molecular External Quality Assessment Programme of the WHO, the Centralized Information System for Infectious Diseases of the WHO EUR and the WHO Measles and Rubella Nucleotide Surveillance databases served as information sources. CONTENT: Molecular proficiency testing conducted by the WHO in 2016 has shown that the expertise for measles and rubella virus genotyping exists in all parts of the EUR. The analysis of surveillance data reported nationally to the WHO in 2013-2016 has revealed some countries with outbreaks but not sufficiently representative molecular data. Long-lasting supranational MV transmission chains were identified. IMPLICATIONS: A more systematic molecular monitoring and recording of the transmission pattern for the whole EUR could help to create a meaningful picture of the elimination process.

A Multibacteriocin Cheese Starter System, Comprising Nisin and Lacticin 3147 in Lactococcus lactis, in Combination with Plantaricin from Lactobacillus plantarum.

Functional starter cultures demonstrating superior technological and food safety properties are advantageous to the food fermentation industry. We evaluated the efficacies of single- and double-bacteriocin-producing starters of Lactococcus lactis capable of producing the class I bacteriocins nisin A and/or lacticin 3147 in terms of starter performance. Single producers were generated by mobilizing the conjugative bacteriophage resistance plasmid pMRC01, carrying lacticin genetic determinants, or the conjugative transposon Tn5276, carrying nisin genetic determinants, to the commercial starter L. lactis CSK2775. The effect of bacteriocin coproduction was examined by superimposing pMRC01 into the newly constructed nisin transconjugant. Transconjugants were improved with regard to antimicrobial activity and bacteriophage insensitivity compared to the recipient strain, and the double producer was immune to both bacteriocins. Bacteriocin production in the starter was stable, although the recipient strain proved to be a more efficient acidifier than transconjugant derivatives. Overall, combinations of class I bacteriocins (the double producer or a combination of single producers) proved to be as effective as individual bacteriocins for controlling Listeria innocua growth in laboratory-scale cheeses. However, using the double producer in combination with the class II bacteriocin producer Lactobacillus plantarum or using the lacticin producer with the class II producer proved to be most effective for reducing bacterial load. As emergence of bacteriocin tolerance was reduced 10-fold in the presence of nisin and lacticin, we suggest that the double producer in conjunction with the class II producer could serve as a protective culture providing a food-grade, multihurdle approach to control pathogenic growth in a variety of industrial applications.IMPORTANCE We generated a suite of single- and double-bacteriocin-producing starter cultures capable of generating the class I bacteriocin lacticin 3147 or nisin or both bacteriocins simultaneously via conjugation. The transconjugants exhibited improved bacteriophage resistance and antimicrobial activity. The single producers proved to be as effective as the double-bacteriocin producer at reducing Listeria numbers in laboratory-scale cheese. However, combining the double producer or the lacticin-producing starter with a class II bacteriocin producer, Lactobacillus plantarum LMG P-26358, proved to be most effective at reducing Listeria numbers and was significantly better than a combination of the three bacteriocin-producing strains, as the double producer is not inhibited by either of the class I bacteriocins. Since the simultaneous use of lacticin and nisin should reduce the emergence of bacteriocin-tolerant derivatives, this study suggests that a protective starter system produced by bacteriocin stacking is a worthwhile multihurdle approach for food safety applications.

Inhibition of Listeria monocytogenes biofilms by bacteriocin-producing bacteria isolated from mushroom substrate.

AIMS: This study was designed to investigate the ability of naturally occurring bacteria isolated from mushroom substrate to prevent biofilm formation by Listeria monocytogenes or to remove existing biofilms in mushroom production facilities. METHODS AND RESULTS: It is generally recognized that L. monocytogenes forms biofilms that can facilitate its survival in food-processing environments. Eleven bacteriocin-producing isolates were identified and the bacteriocins characterized based on heat and enzyme inactivation studies. Further characterization was undertaken by MALDI-TOF mass spectrometry, PCR and sequencing. Production of nisin Z (by Lactococcus lactis isolates), subtilomycin (by Bacillus subtilis isolates) and lichenicidin (by Bacillus licheniformis and Bacillus sonorensis isolates) was detected. In co-culture with L. monocytogenes, the bacteriocin-producing strains could prevent biofilm formation and reduce pre-formed biofilms. CONCLUSIONS: Mushroom substrate can be a source of bacteriocin-producing bacteria that can antagonize L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: The results highlight the potential of bacteriocin-producing strains from mushroom substrate to reduce L. monocytogenes biofilm in food production environments, contributing to a reduction in the risk of food contamination from the environment.

Prediction of individual milk proteins including free amino acids in bovine milk using mid-infrared spectroscopy and their correlations with milk processing characteristics.

The aim of this study was to evaluate the effectiveness of mid-infrared spectroscopy in predicting milk protein and free amino acid (FAA) composition in bovine milk. Milk samples were collected from 7 Irish research herds and represented cows from a range of breeds, parities, and stages of lactation. Mid-infrared spectral data in the range of 900 to 5,000 cm(-1) were available for 730 milk samples; gold standard methods were used to quantify individual protein fractions and FAA of these samples with a view to predicting these gold standard protein fractions and FAA levels with available mid-infrared spectroscopy data. Separate prediction equations were developed for each trait using partial least squares regression; accuracy of prediction was assessed using both cross validation on a calibration data set (n=400 to 591 samples) and external validation on an independent data set (n=143 to 294 samples). The accuracy of prediction in external validation was the same irrespective of whether undertaken on the entire external validation data set or just within the Holstein-Friesian breed. The strongest coefficient of correlation obtained for protein fractions in external validation was 0.74, 0.69, and 0.67 for total casein, total ß-lactoglobulin, and ß-casein, respectively. Total proteins (i.e., total casein, total whey, and total lactoglobulin) were predicted with greater accuracy then their respective component traits; prediction accuracy using the infrared spectrum was superior to prediction using just milk protein concentration. Weak to moderate prediction accuracies were observed for FAA. The greatest coefficient of correlation in both cross validation and external validation was for Gly (0.75), indicating a moderate accuracy of prediction. Overall, the FAA prediction models overpredicted the gold standard values. Near-unity correlations existed between total casein and ß-casein irrespective of whether the traits were based on the gold standard (0.92) or mid-infrared spectroscopy predictions (0.95). Weaker correlations among FAA were observed than the correlations among the protein fractions. Pearson correlations between gold standard protein fractions and the milk processing characteristics of rennet coagulation time, curd firming time, curd firmness, heat coagulating time, pH, and casein micelle size were weak to moderate and ranged from -0.48 (protein and pH) to 0.50 (total casein and a30). Pearson correlations between gold standard FAA and these milk processing characteristics were also weak to moderate and ranged from -0.60 (Val and pH) to 0.49 (Val and K20). Results from this study indicate that mid-infrared spectroscopy has the potential to predict protein fractions and some FAA in milk at a population level.

ET-1 increases reactive oxygen species following hypoxia and high-salt diet in the mouse glomerulus.

AIM: This study was designed to determine whether ET-1 derived from endothelial cells contributes to oxidative stress in the glomerulus of mice subjected to a high-salt diet and/or hypoxia. METHODS: C57BL6/J control mice or vascular endothelial cell ET-1 knockout (VEET KO) mice were subjected to 3-h exposure to hypoxia (8% O2) and/or 2 weeks of high-salt diet (4% NaCl) prior to metabolic cage assessment of renal function and isolation of glomeruli for the determination of reactive oxygen species (ROS). RESULTS: In control mice, hypoxia significantly increased urinary protein excretion during the initial 24 h, but only in animals on a high-salt diet. Hypoxia increased glomerular ET-1 mRNA expression in control, but not in vascular endothelial cell ET-1 knockout (VEET KO) mice. Under normoxic conditions, mice on a high-salt diet had approx. 150% higher glomerular ET-1 mRNA expression compared with a normal-salt diet (P < 0.05). High-salt diet administration significantly increased glomerular ROS production in flox control, but not in glomeruli isolated from VEET KO mice. In C57BL6/J mice, the ETA receptor-selective antagonist, ABT-627, significantly attenuated the increase in glomerular ROS production produced by high-salt diet. In addition, chronic infusion of C57BL6/J mice with a subpressor dose of ET-1 (osmotic pumps) significantly increased the levels of glomerular ROS that were prevented by ETA antagonist treatment. CONCLUSION: These data suggest that both hypoxia and a high-salt diet increase glomerular ROS production via endothelial-derived ET-1-ETA receptor activation and provide a potential mechanism for ET-1-induced nephropathy.

Isolation and characterization of bacteriocin-producing bacteria from the intestinal microbiota of elderly Irish subjects.

AIMS: To isolate and characterize bacteriocins produced by predominant species of lactic acid bacteria from faeces of elderly subjects. METHODS AND RESULTS: Screening over 70,000 colonies, from faecal samples collected from 266 subjects, using the indicator organisms Lactobacillus bulgaricus LMG 6901 and Listeria innocua DPC 3572, identified 55 antimicrobial-producing bacteria. Genomic fingerprinting following ApaI digestion revealed 15 distinct strains. The antimicrobial activities associated with 13 of the 15 strains were sensitive to protease treatment. The predominant antimicrobial-producing species were identified as Lactobacillus salivarius, Lactobacillus gasseri, Lactobacillus acidophilus, Lactobacillus crispatus and Enterococcus spp. A number of previously characterized bacteriocins, including ABP-118 and salivaricin B (from Lact. salivarius), enterocin B (Enterococcus faecium), lactacin B (Lact. acidophilus), gassericin T and a variant of gassericin A (Lact. gasseri), were identified. Interestingly, two antimicrobial-producing species, not generally associated with intestinally derived microorganisms were also isolated: Lactococcus lactis producing nisin Z and Streptococcus mutans producing mutacin II. CONCLUSION: These data suggest that bacteriocin production by intestinal isolates against our chosen targets under the screening conditions used was not frequent (0.08%). SIGNIFICANCE AND IMPACT OF THE STUDY: The results presented are important due to growing evidence indicating bacteriocin production as a potential probiotic trait by virtue of strain dominance and/or pathogen inhibition in the mammalian intestine.

Production of the antimicrobial peptides Caseicin A and B by Bacillus isolates growing on sodium caseinate.

AIMS: The aim of this study was to identify Bacillus isolates capable of degrading sodium caseinate and subsequently to generate bioactive peptides with antimicrobial activity. METHODS AND RESULTS: Sodium caseinate (2.5% w/v) was inoculated separately with 16 Bacillus isolates and allowed to ferment overnight. Protein breakdown in the fermentates was analysed using gel permeation-HPLC (GP-HPLC) and screened for peptides (<3-kDa) with MALDI-TOF mass spectrometry. Caseicin A (IKHQGLPQE) and caseicin B (VLNENLLR), two previously characterized antimicrobial peptides, were identified in the fermentates of both Bacillus cereus and Bacillus thuringiensis isolates. The caseicin peptides were subsequently purified by RP-HPLC and antimicrobial assays indicated that the peptides maintained the previously identified inhibitory activity against the infant formula pathogen Cronobacter sakazakii. CONCLUSIONS: We report a new method using Bacillus sp. to generate two previously characterized antimicrobial peptides from casein. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the potential to exploit Bacillus sp. or the enzymes they produce for the generation of bioactive antimicrobial peptides from bovine casein.

Impact of the broad-spectrum antimicrobial peptide, lacticin 3147, on Streptococcus mutans growing in a biofilm and in human saliva.

AIMS: To evaluate the ability of the broad-spectrum lantibiotic, lacticin 3147, to prevent Streptococcus mutans biofilm formation and disrupt existing biofilms. METHODS AND RESULTS: Minimum inhibitory concentrations (MIC) and minimum biofilm inhibitory concentrations of purified lacticin 3147 were determined using a microdilution method. Lacticin 3147 effectively inhibited planktonic Strep. mutans, with MIC of 1.9-3.8 µmol l(-1). Time-kill kinetic studies confirmed that lacticin 3147 exhibited bactericidal activity against Strep. mutans at 38 µmol l(-1) (or 10× MIC). The effect of lacticin 3147 on biofilm formation and reduction was also determined. Exposure to 6.3-µmol l(-1) lacticin 3147 (2× MIC) resulted in substantial reductions in Strep. mutans biofilm formation while lacticin 3147 was less effective against 1-day-old biofilms. Culture-based analyses revealed that lacticin 3147 (50 µmol l(-1)) significantly inhibited Streptococcus spp. present in human saliva (P < 0.05) with an approximate 4-log reduction in viability compared with the control. CONCLUSIONS: These results indicate that lacticin 3147 may be an effective therapy against Strep. mutans and was shown to substantially attenuate its ability to form a biofilm. SIGNIFICANCE AND IMPACT OF THE STUDY: Lacticin 3147 has the potential to be a useful adjunct to traditional oral therapeutic approaches in addition to its use as a bioactive ingredient for food applications.

Predicting the need for low vision rehabilitation services.

AIMS: To determine the independent predictors of rehabilitation needs for people with low vision using the Impact of Vision Impairment questionnaire (IVI) to measure the quality-of-life consequences of vision-specific restrictions on participation in activities of daily living. METHODS: Patients attending low vision clinics completed the IVI and provided personal and clinical information such as co-morbidities and visual acuity. Rasch analysis was used to generate person measures for the IVI total and three domain scores. Rehabilitation needs were based on "mild", "moderate" or "severe" levels of restriction in participation as determined by the lower, moderate and higher tertiles of persons measures. Logistic regression analyses were used to determine independent predictors of rehabilitation needs. RESULTS: 477 patients (56% women) with a mean age 72 years (SD 15.3) were recruited. Most (74%) had moderate or severe vision loss (presenting visual acuity (VA)<6/18), and 43% had age-related macular degeneration (AMD). Females, shorter duration of vision impairment, having AMD, worse VA, a greater impact of co-morbidities on daily living and reliance on family or friends were univariately associated with poorer IVI scores (p<0.05). In all regression models, VA, the impact of comorbidities on daily living and dependence on family/friends emerged as the three strongest independent predictors of rehabilitation needs. CONCLUSION: In addition to vision, clinicians also need to consider issues relating to dependency when assessing rehabilitation needs. A more holistic approach to patient referral and rehabilitation provision is therefore warranted.

Postcranial skeletal pneumaticity: a case study in the use of quantitative microCT to assess vertebral structure in birds.

Limb elements in birds have been characterized as exhibiting a reduction in trabecular bone, thinner cortices and decreased bending strength when pneumatized, yet it is unclear if these characteristics generalize to the axial skeleton. Thin section techniques, the traditional gold standard for bone structure studies, have most commonly been applied to the study of avian bone. This destructive technique, however, makes it subsequently impossible to use the same samples in experimental testing systems that allow researchers to correlate structure with the mechanical properties of the bone. Micro-computed tomography (microCT), a non-destructive X-ray imaging technique, can be used to assess the effect of pneumatization on vertebral cortical and trabecular bone through virtual extraction and structural quantification of each tissue type. We conducted a preliminary investigation of the application of microCT methods to the study of cortical and trabecular bone structure in a small sample of pneumatic and apneumatic thoracic vertebrae. The sample consisted of two similar-sized anatids, Aix sponsa (n = 7) and Oxyura jamaicensis (n = 5). Volumes of interest were created that contoured (outlined) the boundaries of the ventral cortical bone shell, the trabecular compartment and the whole centrum (cortical bone + trabecular bone), and allowed independent structural analysis of each volume of interest. Results indicated that bone volume fraction of the whole centrum was significantly higher in the apneumatic O. jamaicensis than in the pneumatized A. sponsa (A. sponsa = 36%, O. jamaicensis = 48%, P < 0.05). In contrast, trabecular bone volume fraction was similar between the two species. The ventral cortical bone shell was approximately 23% thinner (P < 0.05) in A. sponsa (0.133 mm) compared with apneumatic O. jamaicensis (0.172 mm). This case study demonstrates that microCT is a powerful non-destructive imaging technique that may be applied to the three-dimensional study of avian bone. The preliminary results suggest that pneumatic and apneumatic vertebrae of comparably sized avian species differ in relative bone volume, with the largest difference apparent at the level of the cortex, and not within trabecular bone. The presence of relatively thin cortices in pneumatic vertebrae is consistent with previous studies contrasting diaphyseal cortical bone between pneumatic and apneumatic long bones. Methodological issues related to this and any comparative microCT study of bone structure are discussed.

RPR-115135, a new non peptidomimetic farnesyltransferase inhibitor, induces G0/G1 arrest only in serum starved cells.

A new non peptidomimetic farnesyltransferase inhibitor, RPR-115135, was studied in an isogenic cell model system consisting of human colon cancer HCT-116 line. HCT-116 cells were transfected with an empty control pCMV vector or with a dominant-negative mutated p53 transgene to disrupt p53 function. Growth inhibitory effects of RPR-115135 were evaluated on cells growing under different conditions (serum starvation, serum starvation and recovery, nocodazole treatment). The cytotoxic activity of RPR-115135 was independent of the cell cycle status of the target cells. Addition of RPR-115135 only to cells exposed to reduced serum conditions (0.1% FCS) resulted in an enhanced ability of HCT-116 cells to arrest in the G0/G1 phase. This arrest response appeared independent of p53/p21cip1/waf-1 function. A reduction of Cyclin A protein amount by RPR-115135 was observed in both clones. These latter results suggest that RPR-115135 might down-regulate the cell cycle factor that would normally impede G0/G1 arrest.

Methods for synchronizing cells at specific stages of the cell cycle.

Exponentially growing cells are asynchronous with respect to the cell cycle stage. Detection of cell cycle-related events is improved by enriching the culture for cells at the stage during which the particular event occurs. Methods for synchronizing cells are provided here, including those based on morphological features of the cell (mitotic shake-off), cellular metabolism (thymidine inhibition, isoleucine depravation), and chemical inhibitors of cell progression in G1 (lovastatin), S (aphidicolin, mimosine), and G2/M (nocodazole). Applications of these methods and the advantages and disadvantages of each are described.

Use of hydrostatic pressure for inactivation of microbial contaminants in cheese.

The objective of this study was to determine the effect of high pressure (HP) on the inactivation of microbial contaminants in Cheddar cheese (Escherichia coli K-12, Staphylococcus aureus ATCC 6538, and Penicillium roqueforti IMI 297987). Initially, cheese slurries inoculated with E. coli, S. aureus, and P. roqueforti were used as a convenient means to define the effects of a range of pressures and temperatures on the viability of these microorganisms. Cheese slurries were subjected to pressures of 50 to 800 MPa for 20 min at temperatures of 10, 20, and 30 degrees C. At 400 MPa, the viability of P. roqueforti in cheese slurry decreased by >2-log-unit cycles at 10 degrees C and by 6-log-unit cycles at temperatures of 20 and 30 degrees C. S. aureus and E. coli were not detected after HP treatments in cheese slurry of >600 MPa at 20 degrees C and >400 MPa at 30 degrees C, respectively. In addition to cell death, the presence of sublethally injured cells in HP-treated slurries was demonstrated by differential plating using nonselective agar incorporating salt or glucose. Kinetic experiments of HP inactivation demonstrated that increasing the pressure from 300 to 400 MPa resulted in a higher degree of inactivation than increasing the pressurization time from 0 to 60 min, indicating a greater antimicrobial impact of pressure. Selected conditions were subsequently tested on Cheddar cheese by adding the isolates to cheese milk and pressure treating the resultant cheeses at 100 to 500 MPa for 20 min at 20 degrees C. The relative sensitivities of the isolates to HP in Cheddar cheese were similar to those observed in the cheese slurry, i.e., P. roqueforti was more sensitive than E. coli, which was more sensitive than S. aureus. The organisms were more sensitive to pressure in cheese than slurry, especially with E. coli. On comparison of the sensitivities of the microorganisms in a pH 5.3 phosphate buffer, cheese slurry, and Cheddar cheese, greatest sensitivity to HP was shown in the pH 5.3 phosphate buffer by S. aureus and P. roqueforti while greatest sensitivity to HP by E. coli was exhibited in Cheddar cheese. Therefore, the medium in which the microorganisms are treated is an important determinant of the level of inactivation observed.

Effects of c-erbB2 overexpression on the drug sensitivities of normal human mammary epithelial cells.

BACKGROUND: Overexpression of the gene c-erbB2, which encodes a receptor tyrosine kinase, in breast tumors has been linked with either increased or decreased response of breast cancer patients to various therapies. In breast cancer cell lines, overexpression of exogenous c-erbB2 sometimes alters drug sensitivities but sometimes has no effect. To avoid the genetic complexities associated with established cancer cell lines, normal human mammary epithelial cells (HMECs) were studied to determine whether c-erbB2 overexpression by itself would alter chemosensitivity. METHODS: HMECs were designed to overexpress c-erbB2, and these cells were then evaluated for alterations in chemosensitivity. RESULTS: HMECs overexpressing c-erbB2 failed to show any alterations in chemosensitivity to a panel of chemotherapeutic agents, as indicated by 95% confidence intervals on growth curves of cells treated with or without the agent of interest. With the use of fluorescence-activated cell sorting to enrich for HMECs overexpressing c-erbB2 on their surface, an 85% pure population of cells was isolated and their chemosensitivity was evaluated. Again, the cells failed to display any alterations in chemosensitivity. CONCLUSIONS: These results suggest that overexpression of c-erbB2 is not sufficient by itself to induce changes in chemosensitivity. Cellular studies using normal human cells in which the complexity of the system can be carefully controlled by the addition of one, two, or even more genes associated with cancer development may provide valuable information about how the products of the genes interact with each other and which combinations are critical in regulating chemosensitivity.

The 1.7 A crystal structure of human cell cycle checkpoint kinase Chk1: implications for Chk1 regulation.

The checkpoint kinase Chk1 is an important mediator of cell cycle arrest following DNA damage. The 1.7 A resolution crystal structures of the human Chk1 kinase domain and its binary complex with an ATP analog has revealed an identical open kinase conformation. The secondary structure and side chain interactions stabilize the activation loop of Chk1 and enable kinase activity without phosphorylation of the catalytic domain. Molecular modeling of the interaction of a Cdc25C peptide with Chk1 has uncovered several conserved residues that are important for substrate selectivity. In addition, we found that the less conserved C-terminal region negatively impacts Chk1 kinase activity.

The Chk1 protein kinase and the Cdc25C regulatory pathways are targets of the anticancer agent UCN-01.

A checkpoint operating in the G(2) phase of the cell cycle prevents entry into mitosis in the presence of DNA damage. UCN-01, a protein kinase inhibitor currently undergoing clinical trials for cancer treatment, abrogates G(2) checkpoint function and sensitizes p53-defective cancer cells to DNA-damaging agents. In most species, the G(2) checkpoint prevents the Cdc25 phosphatase from removing inhibitory phosphate groups from the mitosis-promoting kinase Cdc2. This is accomplished by maintaining Cdc25 in a phosphorylated form that binds 14-3-3 proteins. The checkpoint kinases, Chk1 and Cds1, are proposed to regulate the interactions between human Cdc25C and 14-3-3 proteins by phosphorylating Cdc25C on serine 216. 14-3-3 proteins, in turn, function to keep Cdc25C out of the nucleus. Here we report that UCN-01 caused loss of both serine 216 phosphorylation and 14-3-3 binding to Cdc25C in DNA-damaged cells. In addition, UCN-01 potently inhibited the ability of Chk1 to phosphorylate Cdc25C in vitro. In contrast, Cds1 was refractory to inhibition by UCN-01 in vitro, and Cds1 was still phosphorylated in irradiated cells treated with UCN-01. Thus, neither Cds1 nor kinases upstream of Cds1, such as ataxia telangiectasia-mutated, are targets of UCN-01 action in vivo. Taken together our results identify the Chk1 kinase and the Cdc25C pathway as potential targets of G(2) checkpoint abrogation by UCN-01.

Cellular effects of olomoucine in human lymphoma cells differing in p53 function.

Olomoucine, a purine derivative, inhibits multiple cyclin-dependent kinases that play important roles in regulating the G1/S and G2/M transitions of the cell cycle. In this study we investigated the cellular effects of olomoucine in two human Burkitt's lymphoma cell lines, WMN (containing wild-type p53) and CA46 (containing mutant p53), and found that in consistency with its ability to block the activity of cyclin E/Cdk2 and cyclin B1/Cdc2 kinases, olomoucine caused cell cycle arrest at both G1/S and G2/S boundaries. Moreover, cell cycle arrest occurred equally well in these two cell lines bearing different p53 gene status, suggesting that p53 was not responsible for the cell cycle arrest by olomoucine. A similar p53-independent fashion was also observed in the cytotoxic potency and apoptosis induction of olomoucine, in contrast to ionizing radiation which caused more cytotoxic activity and apoptosis in the WMN cell line bearing wild-type p53 compared with CA46 cells bearing mutant p53. Such p53-independent cytotoxicity of olomoucine was also confirmed in other human Burkitt's lymphoma and lymphoid cell lines containing wild-type and mutant p53. Therefore, our results give an impetus to continued research into olomoucine that might be a very useful chemotherapeutic strategy in the treatment of patients with mutant p53 tumors, at least in lymphoma patients.